Fluorescence immunoassay using polypeptide complex containing fluoro-labeled antibody variable region

Abstract

An object of the present invention is to provide an immunoassay that enables the rapid and convenient quantitative measurement with high detection sensitivity of a target substance in a liquid phase without the need of immobilization and washing steps and enables the visualization of an antigen. The present invention achieves the object by performing sequentially the steps of: (a) bringing, in a liquid phase, a complex comprising a polypeptide containing an antibody light-chain variable region and a polypeptide containing an antibody heavy-chain variable region into contact with an antigen in a specimen for measurement, wherein either or both the polypeptides are labeled with a fluorescent dye(s); (b) detecting the fluorescence of the fluorescent dye(s) or measuring the fluorescence intensity; and (c) calculating the amount of the antigen contained in the sample or visualizing the antigen using the positive correlation between the antigen concentration and the fluorescence intensity of the fluorescent dye(s) as an indicator, thereby measuring the concentration of the target antigen existing in the test substance.

1 . A kit for measuring and/or detecting the concentration of an antigen, which is characterized by enabling the measurement of the concentration of an antigen or the visualization of an antigen using a positive correlation between the concentration of an antigen and the fluorescence intensity of a fluorescent dye in a liquid phase, as an indicator, wherein, either or both a polypeptide containing an antibody light chain variable region and a polypeptide containing an antibody heavy chain variable region are labeled with a fluorescent dye, and the kit is provided with a complex comprising the polypeptide containing the antibody light chain variable region and the polypeptide containing the antibody heavy chain variable region. 
     
     
         2 . The kit for measuring and/or detecting the concentration of an antigen according to  claim 1 , which is characterized in that the polypeptide containing the antibody light chain variable region and the polypeptide containing the antibody heavy chain variable region are each labeled with the same fluorescent dye. 
     
     
         3 . The kit for measuring and/or detecting the concentration of an antigen according to  claim 1 , which is characterized in that the polypeptide containing the antibody light chain variable region and the polypeptide containing the antibody heavy chain variable region are each labeled with a different type of fluorescent dye. 
     
     
         4 . The kit for measuring and/or detecting the concentration of an antigen according to  claim 1 , which is characterized in that either the polypeptide containing the antibody light chain variable region or the polypeptide containing the antibody heavy chain variable region is labeled with a fluorescent dye and the other is labeled with a quencher for quenching the fluorescent dye. 
     
     
         5 . The kit for measuring and/or detecting the concentration of an antigen according to  claim 1 , which is characterized in that either the polypeptide containing the antibody light chain variable region or the polypeptide containing the antibody heavy chain variable region is labeled with a fluorescent dye. 
     
     
         6 . The kit for measuring and/or detecting the concentration of an antigen according to  claim 1 , which is characterized in that a complex comprising the polypeptide containing the antibody light chain variable region and the polypeptide containing the antibody heavy chain variable region is a Fab (Fragment, antigen binding). 
     
     
         7 . The kit for measuring and/or detecting the concentration of an antigen according to  claim 1 , which is characterized in that the fluorescent dye is selected from a rhodamine-based fluorescent dye and an oxazine-based fluorescent dye. 
     
     
         8 . The kit for measuring and/or detecting the concentration of an antigen according to  claim 7 , which is characterized in that the fluorescent dye is selected from carboxy rhodamine 110, carboxytetramethyl rhodamine, and ATTO 655 (trade name). 
     
     
         9 . The kit for measuring and/or detecting the concentration of an antigen according to  claim 4 , which is characterized in that the quencher is 7-nitrobenzofurazan (NBD). 
     
     
         10 . A method for measuring and/or detecting the concentration of an antigen, which is characterized by comprising the following steps (a) to (c) in sequence,
 (a) a step of bringing, in a liquid phase, a complex into contact with an antigen in a specimen for measurement, wherein the complex comprises a polypeptide containing an antibody light chain variable region and a polypeptide containing an antibody heavy chain variable region, in which either or both polypeptides are labeled with a fluorescent dye;   (b) a step of detecting the fluorescence of the fluorescent dye, or measuring the fluorescence intensity of the fluorescent dye; and   (c) a step of calculating the amount of the antigen contained in a sample or visualizing the antigen, using a positive correlation between the concentration of the antigen and the fluorescence intensity of the fluorescent dye as an indicator.   
     
     
         11 . The method for measuring and/or detecting the concentration of an antigen according to  claim 10 , which is characterized in that the polypeptide containing the antibody light chain variable region and the polypeptide containing the antibody heavy chain variable region are each labeled with the same fluorescent dye. 
     
     
         12 . The method for measuring and/or detecting the concentration of an antigen according to  claim 10 , which is characterized in that the polypeptide containing the antibody light chain variable region and the polypeptide containing the antibody heavy chain variable region are each labeled with a different type of fluorescent dye. 
     
     
         13 . The method for measuring and/or detecting the concentration of an antigen according to  claim 10 , which is characterized in that either the polypeptide containing the antibody light chain variable region or the polypeptide containing the antibody heavy chain variable region is labeled with a fluorescent dye, and the other is labeled with a quencher for quenching the fluorescent dye. 
     
     
         14 . The method for measuring and/or detecting the concentration of an antigen according to  claim 10 , which is characterized in that either the polypeptide containing the antibody light chain variable region or the polypeptide containing the antibody heavy chain variable region is labeled with a fluorescent dye. 
     
     
         15 . The method for measuring and/or detecting the concentration of an antigen according to  claim 10 , which is characterized in that the complex comprising the polypeptide that contains the antibody light chain variable region and the polypeptide that contains the antibody heavy chain variable region is a Fab (Fragment, antigen binding). 
     
     
         16 . The method for measuring and/or detecting the concentration of an antigen according to  claim 10 , which is characterized in that the antigen is a low molecular weight compound. 
     
     
         17 . The method for measuring and/or detecting the concentration of an antigen according to  claim 10 , which is characterized in that the antigen is human osteocalcin, bisphenol A, serum albumin, clenbuterol, ractopamine, cotinine, influenza A virus hemagglutinin, a morphine, a methamphetamine, cocaine, tetrahydrocannabinol, or ketamine.